Macrophages and dendritic cells (DCs) play key roles in HIV-1 infection and in innate immunity, but the role of innate immunity, and in particular TLRs, in HIV-1 infection and disease progression is poorly understood. LPS and poly(I:C) (TLR4 and TLR3 ligands, respectively), have been shown to inhibit HIV-1 replication in and infection of macrophages, albeit the mechanisms are unclear. We have found that stimulation of human monocyte-derived macrophages (MDMs) through TLR3 and 4, but not through TLR7 and 9, abrogates their susceptibility to HIV-1 infection. TLR3-mediated suppression of infection was also observed in monocyte derived DCs (moDCs). Subsequent studies in MDMs showed no change in surface expression of CD4 and CCR5 between TLR3/4 and TLR7/9-stimulated cells, similar production of type I interferons, pro-inflammatory cytokines and beta-chemokines, and the same effect on susceptibility to infection in stimulated cells exposed to vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped virus. MicroRNAs (miRNAs) are potent regulators of gene expression, playing pivotal roles in development, differentiation and in the regulation of immune and inflammatory responses. It has been shown that TLR stimulation can modulate miRNA expression, and some miRNAs seem to be involved in the modulation of susceptibility to HIV-1 infection in primary cells. In this sense, we have found that TLR3-, 4- and 7-stimulated MDMs display overlapping but distinct changes in miRNA expression profiles, and we have identified a subset of four miRNAs that are up-regulated in cells stimulated through TLR3 and 4 but not through TLR7. Among them, miR-155 showed the highest and more consistent up-regulation, and we have since confirmed its specific up-regulation in TLR3/4-stimulated MDMs and in TLR3-stimulated moDCs from multiple donors. In addition, pre-treatment of poly(I:C)-stimulated MDMs with an anti-miR-155 antagomir inhibited miR-155 induction and increased their susceptibility to HIV-1 infection. Moreover, miR-155 over-expression in MDMs and cell lines led to suppression or reduction of HIV-1 infection. Target analysis has suggested that several confirmed or potential HIV-dependency factors (HDFs: several importins and nucleoporins, and LEDGF, among others), are predicted targets for miR-155, and reduced mRNA levels in selected potential targets have been observed. Thus, we hypothesize that miR-155 has the capacity to reduce susceptibility to HIV-1 infection by decreasing the levels of one or more HDFs, leading to an intracellular environment that is less conducive to productive HIV-1 infection. Our specific aims are: (i) To identify and validate mRNA targets for miR-155 that could participate in the modulation of susceptibility to HIV-1 infection in macrophages and dendritic cells; and (ii) To define the mechanism(s) by which modulation of miR-155 levels leads to altered susceptibility to HIV-1 infection in MDMs and moDCs. These novel studies will shed light on our limited understanding of the interplay between miRNAs and HIV-1, and might open new avenues to explore the use of miR-155 to decrease susceptibility to HIV-1 infection. PUBLIC HEALTH RELEVANCE: Our innovative studies will define the relationship between stimulation of innate immunity through selected Toll-like receptors (TLR), altered expression of microRNA-155 and differential susceptibility of macrophages and dendritic cells to HIV-1 infection. This research will shed light on our current limited understanding of the interplay between the host's innate immunity and HIV-1. In addition, it could open new avenues to explore the potential usefulness of microRNA modulation, and specifically microRNA-155, for the prevention of HIV-1 infection.